The present invention is directed to compounds which act as prodrugs of AICA riboside and certain analogs of it. AICA riboside monophosphate is a naturally occurring intermediate in purine biosynthesis. AICA riboside is also naturally occurring and is now known to enable adenosine release from cells during net ATP catabolism. By virtue of its adenosine releasing abilities, AICA riboside has many therapeutic uses. However, we have discovered that AICA riboside does not cross the blood-brain barrier well and is inefficiently absorbed from the gastrointestinal tract; both characteristics decrease its full potential for use as a therapeutic agent.
We have also discovered that AICA riboside, and AICA riboside pro-drugs and analogs can be used to lower blood glucose levels in animals, including rats, rabbits, dogs and man. These compounds are surprisingly efficacious for lowering blood sugar and are believed to be partially causing their effect by decreasing hepatic gluconeogenesis. These compounds will be useful for the treatment of animals for conditions including hyperglycemia, insulin resistance, insulin deficiency, diabetes mellitis, Syndrome X, to control the hyperglycemia and/or hyperlipidemia associated with total parenteral nutrition, or a combination of these effects. While AICA riboside does not have the enhanced bioavailability as described for those pro-drugs set forth herein as useful for penetrating the gut barrier, it may be nevertheless useful for the above conditions because AICA riboside itself will be present in amounts sufficient to reach the liver, as we have also discovered. AICA riboside monophosphate is implicated by our studies to be the causative agent and, accordingly, it and monophosphate forms of prodrug and analog compounds noted herein are within the scope of our invention.
Adenosine, 9-.beta.-D-ribofuranosyladenine (the nucleoside of the purine adenine), belongs to the class of biochemicals termed purine nucleosides and is a key biochemical cell regulatory molecule, as described by Fox and Kelly in the Annual Reviews of Biochemistry, Vol. 47, p. 635, 1978.
Adenosine interacts with a wide variety of cell types and is responsible for a myriad of biological effects. Adenosine serves a major role in brain as an inhibitory neuromodulator (see Snyder, S. H., Ann. Rev. Neural Sci. 8:103-124 1985, Marangos, et al., NeuroSci and Biobehav. Rev. 9:421-430 (1985), Dunwiddie, Int. Rev. Neurobiol., 27:63-130 (1985)). This action is mediated by ectocellular receptors (Londos et al., Regulatory Functions of Adenosine, pp. 17-32 (Berne et al., ed.) (1983)). Among the documented actions of adenosine on nervous tissue are the inhibition of neural firing (Phillis et al., Europ. J. Pharmacol., 30:125-129 (1975)) and of calcium dependent neurotransmitter release (Dunwiddie, 1985). Behaviorally, adenosine and its metabolically stable analogs have profound anticonvulsant and sedative effects (Dunwiddie et al., J. Pharmacol. and Exptl. Therapeut., 220:70-76 (1982); Radulovacki et al., J. Pharmacol. Exptl. Thera., 228:268-274 (1981)) that are effectively reversed by specific adenosine receptor antagonists. In fact, adenosine has been proposed to serve as a natural anticonvulsant, and agents that alter its extracellular levels are modulators of seizure activity (Dragunow et al., Epilepsia 26:480-487 (1985); Lee et al., Brain Res., 21:1650-164 (1984)). In addition, adenosine is a potent vasodilator, an inhibitor of immune cell function, an inhibitor of granulocyte oxygen free radical production, an anti-arrhythmic, and an inhibitory neuromodulator. Given its broad spectrum of biological activity, considerable effort has been aimed at establishing practical therapeutic uses for adenosine and its analogs.
Since adenosine is thought to act at the level of the cell plasma membrane by binding to receptors anchored in the membrane, past work has included attempts to increase extra-cellular levels of adenosine by administering it into the blood stream. Unfortunately, because adenosine is toxic at concentrations that have to be administered to a patient to maintain an efficacious extracellular therapeutic level, the administration of adenosine alone is of limited therapeutic use. Further, adenosine receptors are subject to negative feedback control following exposure to adenosine, including down-regulation of the receptors.
Other ways of achieving the effect of a high local extracellular level of adenosine exist and have also been studied. They include: a) interference with the uptake of adenosine with reagents that specifically block adenosine transport, as described by Paterson et al., in the Annals of the New York Academy of Sciences, Vol. 255, p. 402 (1975); b) prevention of the degradation of adenosine, as described by Carson and Seegmiller in The Journal of Clinical Investigation, Vol. 57, p. 274 (1976); and c) the use of analogs of adenosine constructed to bind to adenosine cell plasma membrane receptors.
There are a large repertoire of chemicals that can inhibit the cellular uptake of adenosine. Some do so specifically, and are essentially competitive inhibitors of adenosine uptake, and others inhibit nonspecifically. P-nitrobenzylthioinosine and dipyridamole appear to be competitive inhibitors. A variety of other chemicals, including colchicine, phenethyalcohol and papaverine inhibit uptake nonspecifically.
Extracellular levels of adenosine can be increased by the use of chemicals that inhibit enzymatic degradation of adenosine. Previous work has focused on identifying inhibitors of adenosine deaminase, which participates in the conversion of adenosine to inosine. Adenosine deaminase activity is inhibited by coformycin, 2'-deoxycoformycin, and erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride.
A number of adenosine receptor agonists and antagonists have been generated having structural modifications in the purine ring, alterations in substituent groups attached to the purine ring, and modifications or alterations in the carbohydrate moiety. Halogenated adenosine derivatives appear to have been promising as agonists or antagonists and, as described by Wolff et al. in the Journal of Biological Chemistry, Vol. 252, p. 681, 1977, exert biological effects in experimental systems similar to those caused by adenosine. Derivatives with N-6 or 5'-substitutions have also shown promise.
Although all three techniques discussed above may have advantages over the use of adenosine alone, they have been found to have several disadvantages. The major disadvantages of these techniques are that they rely on chemicals that have adverse side effects, primarily due to the fact that they must be administered in doses that are toxic, and that they affect most cell types nonselectively. As described in Purine Metabolism in Man, (eds. De Baryn, Simmonds and Muller), Plenum Press, New York, 1984, most cells in the body carry receptors for adenosine. Consequently the use of techniques that increase adenosine levels generally throughout the body can cause unwanted, dramatic changes in normal cellular physiology. In addition, adenosine deaminase inhibitors prevent the degradation of deoxyadenosine which is a potent immunotoxin. (Gruber et al., Ann. New York Acad. Sci. 451:315-318 (1985)).
It will be appreciated that compounds which increase extracellular levels of adenosine or adenosine analogs at specific times during a pathologic event, without complex side effects, and which would permit increased adenosine levels to be selectively targeted to cells that would benefit most from them, would be of considerable therapeutic use. By way of example, such compounds would be especially useful in the prevention of, or response during, an ischemic event, such as heart attack or stroke, or other event involving an undesired restricted or decreased blood flow, such as atherosclerosis or skin flap surgery, for adenosine is a vasodilator and prevents the production of superoxide radicals by granulocytes. Such compounds would also be useful in the prophylactic or affirmative treatment of pathologic states involving increased cellular excitation, such as (1) seizures or epilepsy, (2) arrhythmias (3) inflammation due to, for example, arthritis, autoimmune disease, Adult Respiratory Distress Syndrome (ARDS), and granulocyte activation by complement from blood contact with artificial membranes as occurs during dialysis or with heart-lung machines. It would further be useful in the treatment of patients who might have chronic low adenosine such as those suffering from autism, cerebral palsy, insomnia and other neuropsychiatric symptoms, including schizophrenia. The compounds useful in the invention may be used to accomplish these ends.
Clearly, there is a need for more effective anticonvulsant therapeutic compounds and strategies since most of the currently used antiseizure agents are toxic (e.g., dilantin), or are without efficacy in many patients. Adenosine releasing agents, which enhance adenosine levels during net ATP catabolism will be useful for the treatment of seizure disorders.
Compounds which selectively increase extracellular adenosine will also be used in the prophylactic protection of cells in the hippocampus implicated in memory. The hippocampus has more adenosine and glutamate receptors than any other area of the brain. Accordingly, as described below, it is most sensitive to stroke or any condition of low blood flow to the brain. Some recent studies support the theory that Alzheimer's disease may result from chronic subclinical cerebral ischemia. The compounds of the invention will be used for the treatment and/or prevention of both overt stroke and Alzheimer's disease.
It is now established that relatively short periods of brain ischemia (on the order of 2 to 8 minutes) set into motion a series of events that lead to an eventual death of selected neuronal populations in brain. This process is called delayed excitotoxicity and it is caused by the ischemia-induced release of the excitatory amino acid (EAA) neurotransmitters glutamate and aspartate. Within several days post-stroke the neurons in the brain are overstimulated by EAA's to the point of metabolic exhaustion and death. Because glutamate appears to be the major factor involved in post-stroke cell damage, the blockade of glutamate receptors in brain could be beneficial in stroke therapy. In animals, glutamate receptor blockers have been shown to be effective in alleviating or reversing stroke associated neural damage. These receptor blockers have, however, been shown to lack specificity and produce many undesirable side effects. Church, et al., "Excitatory Amino Acid Transmission," pp. 115-118 (Alan R. Liss, Inc. 1987).
Adenosine has been shown to be a potent inhibitor of glutamate release in brain. The CA-1 region of brain is selectively sensitive to post-stroke destruction. In studies, where observations were made at one, three and six days post-stroke the CA-1 area was shown to be progressively destroyed over time. However, where cyclohexyladenosine ("CHA") a global adenosine agonist, was given shortly after the stroke, the CA-1 area was markedly protected. (Daval et al., Brain Res. 491: 212-226 (1989).) That beneficial effect was also seen in the survival rate of the animals. Because of its global effect, however, CHA has non-specific side effects. For example it undesirably will lower blood pressure and could remove blood from the ischemic area, thereby causing further decreased blood flow.
The compounds of the invention described and claimed herein not only show the beneficial adenosine release (glutamate inhibiting properties) but are both site and event specific, avoiding the unwanted global action of known adenosine agonists. These compounds will also be used in the treatment of neurodegenerative diseases related to the exaggerated action of excitatory amino acids, such as Parkinson's disease.
Another area of medical importance is the treatment of neurological diseases or conditions arising from elevated levels of homocysteine (e.g., vitamin B12 deficiencies). The novel AICA riboside prodrugs of this invention may be used for such purposes as well.
A further area of medical importance is the treatment of allergic diseases, which can be accomplished by either preventing mast cell activation, or by interfering with the mediators of allergic responses which are secreted by mast cells. Mast cell activation can be down-regulated by immunotherapy (allergy shots) or by mast cell stabilizers such as cromalyn sodium, corticosteroids and aminophylline. There are also therapeutic agents which interfere with the products of mast cells such as anti-histamines and adrenergic agents. The mechanism of action of mast cell stabilization is not clearly understood. In the case of aminophylline it is possible that it acts as an adenosine receptor antagonist. However, agents such as cromalyn sodium and the corticosteroids are not as well understood.
It will be appreciated, therefore, that effective allergy treatment with compounds which will not show any of the side effects of the above noted compounds, such as drowsiness in the case of the anti-histamines, agitation in the case of adrenergic agents, and Cushing disease symptoms in the case of the corticosteroids would be of great significance and utility. In contrast to compounds useful in the present invention, the AICA riboside prodrugs, none of the three known mast cell stabilizers are known or believed to be metabolized in the cell to purine nucleoside triphosphates or purine nucleoside monophosphates.
The use of AICA riboside and prodrugs of AICA riboside as antiviral agents and for increasing the antiviral activity of AZT is disclosed in commonly-assigned U.S. patent application Ser. No. 301,454, "Antivirals and Methods for Increasing the Antiviral Activity of AZT", filed Jan. 24, 1989, the disclosure of which is incorporated herein by reference.
Certain derivatives of AICA riboside have been prepared and used as intermediates in the synthesis of nucleosides such as adenosine or nucleoside analogs such as 3'-deoxy-thio-AICA riboside. See, e.g., U.S. Pat. No. 3,450,693 to Suzuki et al.; Miyoshi et al., Chem. Pharm. Bull. 24(9): 2089-2093 (1976); Chambers et al., Nucleosides & Nucleotides 7(3): 339-346 (1988); Srivastava, J. Org. Chem. 40(20): 2920-2924 (1975).
Hyperglycemia has been reported to be associated with a poor prognosis for stroke. (Helgason, Stroke 19(8): 1049-1053 (1988). In addition, mild hypoglycemia induced by insulin treatment has been shown to improve survival and morbidity from experimentally induced infarct. (LeMay et al., Stroke 19(11): 1411-1419 (1988)). We believe that AICA riboside and the prodrugs of the present invention will be useful to help protect against ischemic injury to the central nervous system (CNS) at least partly by their ability to lower blood glucose.
Hyperglycemia and related diabetic conditions are generally divided into "type I" or severe (typically insulin requiring) and "type II" or mild (typically controlled by oral hypoglycemic agents and/or diet and exercise). Type I diabetic patients have severe insulin deficiency with complications typically including hyperglycemia and ketoacidosis. Type II diabetic patients typically have milder insulin deficiency or decreased insulin sensitivity associated with hyperglycemia predominantly from accelerated hepatic gluconeogenesis. Both forms of diabetic conditions are associated with atherosclerosis and ischemic organ injury.
Oral hypoglycemic agents that are currently available clinically include sulfonylureas (e.g., tolbutamide, tolazamide, acetohexamide, chlorpropamide, glyburide, glipizide) and biguanides (e.g. phenformin and metformin). The sulfonylurea class of drugs lower blood sugar acutely in man and experimental animals by causing insulin release but in long term studies, their activity appears to involve extra pancreatic effects. These drugs are active on potassium cation channels, but it is not known if this activity is related to their hypoglycemic effects. The sulfonyl-urea class of drugs are not ideal hypoglycemic agents for a variety of reasons; moreover, they have been associated with increased risk of cardiovascular disease and can be of insufficient efficacy for many Type II diabetes patients.
The biguanide class of drugs reduce blood sugar by increasing peripheral utilization of glucose and by decreasing hepatic glucose production, both effects presumably caused by inhibiting oxidative phosphorylation. In addition, because of their inhibition of oxidative phosphorylation, the biguanides have been associated with fatal lactic acidosis and, for that reason, are at present not available clinically in the United States.
Other compounds which lower blood sugar have been described in the literature, but none of them is available clinically due to other toxicities. (See Sherratt, H. S. A., "Inhibition of Gluconeogenesis by Non-Hormonal Hypoglycaemic Compounds" in Short-Term Regulation of Liver Metabolism, pp. 199-277 (Hue, L. and Van de Werve, G., ed.s, Elsovier/North Holland Biomedical Press, 1981)). D-Ribose has been reported to cause hypoglycemia after oral or intravenous administration to experimental animals and humans and Foley (J. Clin. Invest. 37: 719-735 (1958)) demonstrated an inhibition of phosphoglucomutase by ribose-5'-phosphate (formed intracellularly after ribose therapy). Although others have suggested that ribose lowers glucose via increased insulin release (Ishiwita et al., Endoncinol. Japan 25: 163-169 (1978)), the preponderance of evidence favors decreased glucose production over increased insulin release.
Fructose diphosphatase has been suggested as an ideal target for new hypoglycemic agents, since it is one of two control steps in gluconeogenesis. (See, Sherratt, supra 1981) However, therapeutic agents which lower its activity are not presently clinically available. Fructose diphosphatase is inhibited by AMP and activated by ATP, being responsive to the cellular energy charge. Pyruvate carboxylase, the other major regulatory step in gluconeogenesis, is the first committed step towards glucose production and is regulated by the availability of acetyl CoA; however, its inhibition would result in interruption of mitochondrial function.
The present invention is directed to purine prodrugs and analogs which exhibit and, in some cases improve upon, the positive biological effects of AICA riboside and other adenosine releasing compounds without the negative effects of systemic adenosine. The compounds herein defined may be used as prodrugs. The novel compounds typically exhibit one or more of the following improvements over AICA riboside: 1) more potent adenosine releasing effects; 2) increased half-lives; 3) increased brain penetration; 4) increased oral bioavailability; 5) increased myocardial targeting; 6) in some cases efficacy improvements over AICA riboside itself.
The AICA riboside prodrugs of this invention may be used in treatment and prevention of a number of disorders, some of which already have been mentioned.